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PubMed2004

Three-dimensional structure of C complex spliceosomes by electron microscopy.

The spliceosome is a multimegadalton RNA-protein machine that removes noncoding sequences from nascent pre-mRNAs. Recruitment of the spliceosome to splice sites and subsequent splicing require a series of dynamic interactions among the spliceosome's component U snRNPs and many additional protein factors. These dynamics present several challenges for structural analyses, including purification of stable complexes to compositional homogeneity and assessment of conformational heterogeneity. We have isolated spliceosomes arrested before the second chemical step of splicing (C complex) in which U2, U5 and U6 snRNAs are stably associated. Using electron microscopy, we obtained images of C complex spliceosomes under cryogenic conditions and determined a three-dimensional structure of a core complex to a resolution of 30 A. The structure reveals a particle of dimensions 27 x 22 x 24 nm with a relatively open arrangement of three primary domains.

Cryoelectron Microscopy↗

PubMed2003

Reverse genetics for crimean-congo hemorrhagic fever virus.

The widespread geographical distribution of Crimean-Congo hemorrhagic fever (CCHF) virus (more than 30 countries) and its ability to produce severe human disease with high mortality rates (up to 60%) make CCHF a major public health concern worldwide. We describe here the successful establishment of a reverse genetics technology for CCHF virus, a member of the genus Nairovirus, family BUNYAVIRIDAE: The RNA polymerase I (pol I) system was used to generate artificial viral RNA genome segments (minigenomes), which contained different reporter genes in antisense (virus RNA) or sense (virus-complementary RNA) orientation flanked by the noncoding regions of the CCHF virus S segment. Reporter gene expression was observed in different eukaryotic cell lines following transfection and subsequent superinfection with CCHF virus, confirming encapsidation, transcription, and replication of the pol I-derived minigenomes. The successful transfer of reporter gene activity to fresh cells demonstrated the generation of recombinant CCHF viruses, thereby confirming the packaging of the pol I-derived minigenomes into progeny viruses. The system offers a unique opportunity to study the biology of nairoviruses and to develop therapeutic and prophylactic measures against CCHF infections. In addition, we demonstrated for the first time that the human pol I system can be used to develop reverse genetics approaches for viruses in the family BUNYAVIRIDAE: This is important since it might facilitate the manipulation of bunyaviruses with cell and host tropisms restricted to primates.

Animals↗

PubMed1996

In vitro transcription and translation inhibition by anti-promyelocytic leukemia (PML)/retinoic acid receptor alpha and anti-PML peptide nucleic acid.

Peptide nucleic acids (PNAs) complementary to the 15 bases around the fusion point of both genomic DNA and cDNA of the promyelocytic leukemia/retinoic acid receptor alpha (PML/ RAR alpha; P/R) hybrid gene present in acute promyelocytic leukemia cells were synthesized and shown by gel retardation experiments to specifically bind oligonucleotides corresponding to the fusion region of the P/R molecule. PNA was also able to successfully compete with anti-P/R DNA for duplex formation with P/R DNA and to displace the anti-P/R DNA from dsDNA. In vitro transcribed P/R RNA from two inserts of approximately 350 to approximately 700 bp were tested in gel acceleration experiments with fluorescein-conjugated PNA and showed stable binding (resistant to denaturing conditions) of PNA to the newly transcribed RNA. Control RNA or transcripts from the noncoding strand did not bind PNA. However, this PNA, although able to specifically clamp polymerase chain reaction, was incapable of inhibiting in vitro translation of the PML/RAR alpha mRNA, even when a bis-PNA was used. Therefore, a PNA was targeted against the start region of the P/R cDNA and against poly-purine regions of the gene. Specific inhibition of in vitro translation and transcription was shown, starting at concentrations as low as 100 nmol/L. When oligonucleotides presenting the same sequence were compared, PNA proved to be approximately 40 times more active. In conclusion, in vitro inhibition of translation and transcription of the P/R gene can be obtained with PNA; however, it is still necessary to target the ATG start region or poly-purine regions of the gene.

Base Sequence↗

PubMed1984

Nucleotide sequence 5' of the chicken c-myc coding region: localization of a noncoding exon that is absent from myc transcripts in most avian leukosis virus-induced lymphomas.

We have determined the nucleotide sequence of the 2.2-kilobase-pair region upstream of the chicken c-myc coding exons. Using RNA blot analysis, we have localized a noncoding exon to a region that is separated from the c-myc coding sequences by an intron of 700-800 base pairs. In most avian leukosis virus-induced lymphomas proviral integration has occurred within, or downstream of, the first exon, thus presumably displacing the regulatory sequences that normally control c-myc expression. More than 70% of the integration sites were clustered in a 250-base-pair region in the first intron, immediately preceding the coding sequences. Sequences from the upstream noncoding exon were absent from the myc transcripts in these lymphomas; RNA transcripts from the normal c-myc allele were not expressed at detectable levels.

Animals↗

PubMed1988

Translation in mammalian cells of a gene linked to the poliovirus 5' noncoding region.

The central portion (region P) of the 742-nucleotide noncoding 5' end of poliovirus allows the RNA to initiate protein synthesis in the absence of the usual 5' 7-methylguanosine capping group. Poliovirus 5' noncoding region was fused to a reporter gene and transfected into cells. There was extensive augmentation of the expression of this gene by poliovirus-mediated inhibition of cap-dependent protein synthesis. That the construct initiated in a cap-independent manner was verified through in vitro experiments. Small lesions throughout region P blocked its initiation function, implying that a coherent functional unit, hundreds of nucleotides long, is responsible for cap-independent initiation by poliovirus RNA.

Acetyltransferases↗

PubMed1988

Deletion of the N-terminal region of Xenopus transcription factor IIIA inhibits specific binding to the 5 S RNA gene.

Xenopus transcription factor IIIA (TFIIIA) is expressed in Escherichia coli by utilizing one plasmid with a T7 RNA polymerase gene and another plasmid with TFIIIA cDNA cloned downstream of a T7 promoter. Wild-type TFIIIA and a TFIIIA deletion mutant, isolated from E. coli cell extracts, are identified by antiserum against native TFIIIA purified from Xenopus immature oocytes. DNase I protection experiments indicate that wild-type TFIIIA, synthesized from a full-length TFIIIA cDNA, binds specifically to the coding and noncoding strands of the 5 S RNA gene. The TFIIIA deletion mutant, expressed from TFIIIA cDNA lacking the coding sequence for the N-terminal 29 amino acids, fails to bind specifically to the 5 S RNA gene as judged by its inability to protect to any degree the coding or noncoding strands of the gene from DNase I digestion. Both wild-type TFIIIA and the N-terminal deletion mutant promote DNA renaturation.

Animals↗

PubMed2003

High expression of B-cell receptor inducible gene BIC in all subtypes of Hodgkin lymphoma.

In a search for genes specifically expressed in Reed-Sternberg (RS) cells of Hodgkin lymphoma (HL), we applied the serial analysis of gene expression (SAGE) technique on the HL-derived cell line DEV. Genes highly expressed in DEV were subjected to an RT-PCR analysis to confirm the SAGE results. For one of the genes, a high expression was observed in DEV and other HL-derived cell lines but not in non-Hodgkin lymphoma (NHL)-derived cell lines and normal controls, suggesting an HL-specific expression. This gene corresponds to the human BIC gene, a member of the noncoding mRNA-like molecules. RNA in situ hybridization (ISH) indicated an exclusive nucleolar localization of BIC transcripts in all RS cells in 91% of HL cases, including nodular lymphocyte predominance (NLP) HL and classical HL. Analyses of normal human tissues revealed BIC transcripts in only a small number of CD20-positive B-cells in lymph node and tonsil tissue, albeit at a much lower level compared to that of RS cells. BIC RT-PCR in the Burkitt lymphoma-derived cell line Ramos demonstrated a significant up-regulation upon cross-linking of the B-cell receptor (BcR). IkappaBalpha-mediated blocking of NF-kappaB translocation in Ramos did not effect the up-regulation of BIC expression upon BcR triggering, suggesting that activation of NF-kappaB is not involved in regulation of BIC expression. In summary, our data show that expression of BIC is specific for RS cells of HL. In normal tissue, BIC is expressed weakly in a minority of germinal center B cells. Expression of BIC can be modified/influenced by BcR triggering, indicating that BIC might play a role in the selection of B cells.

Animals↗

PubMed1998

Functional significance of three basic N-terminal amino acids of alfalfa mosaic virus coat protein.

Infection of tobacco protoplasts with mutant alfalfa mosaic virus (AMV) RNAs indicated that three basic amino acids in the N-terminus of AMV coat protein are important for the biological activity of the coat protein in the beginning of infection. Substitution of alanines for lysines at position 14 or 17 in the coat protein resulted in a 5- or 10-fold reduction in the activity of the protein, respectively. However, substitution of alanine for arginine at position 18 entirely abolished activity. Arginine 18 was also required for the coat protein to bind to the 3' noncoding region of the virus RNA in vitro, whereas lysine 14 or 17 was not required. Thus, these results indicate that arginine 18 is essential for the activity of the coat protein in early infection and that binding of the coat protein to AMV RNA correlates with activity.

Alfalfa mosaic virus↗

PubMed1994

Interaction of cellular proteins with the poliovirus 5' noncoding region.

The 5' noncoding region (NCR) of poliovirus RNA is folded into a complex structure comprised of multiple, critically spaced, stem-loop domains. Mutations in at least one of these domains markedly affects the neurovirulence of the virus. Two proteins have been identified recently which bind and apparently mediate functions of the 5' NCR in translation. We have demonstrated specific binding of three additional proteins in a Hela cell ribosomal salt wash that can be crosslinked to specific stem-loop segments of the 5' NCR. These same RNA segments inhibit translation of polio RNA in vitro, presumably by competing for protein binding. The Sabin vaccine strain of polio RNA exhibits a reduced affinity of binding for specific proteins. The determinant for this reduction appears to be a single nucleotide difference at position 480 between the neurovirulent and attenuated viral strains.

Binding, Competitive↗

PubMed1994

Analytical expression of the purine/pyrimidine autocorrelation function after and before random mutations.

The mutation process is a classical evolutionary genetic process. The type of mutations studied here is the random substitutions of a purine base R (adenine or guanine) by a pyrimidine base Y (cytosine or thymine) and reciprocally (transversions). The analytical expressions derived allow us to analyze in genes the occurrence probabilities of motifs and d-motifs (two motifs separated by any d bases) on the R/Y alphabet under transversions. These motif probabilities can be obtained after transversions (in the evolutionary sense; from the past to the present) and, unexpectedly, also before transversions (after back transversions, in the inverse evolutionary sense, from the present to the past). This theoretical part in Section 2 is a first generalization of a particular formula recently derived. The application in Section 3 is based on the analytical expression giving the autocorrelation function (the d-motif probabilities) before transversions. It allows us to study primitive genes from actual genes. This approach solves a biological problem. The protein coding genes of chloroplasts and mitochondria have a preferential occurrence of the 6-motif YRY(N)6YRY (maximum of the autocorrelation function for d = 6, N = R or Y) with a periodicity modulo 3. The YRY(N)6YRY preferential occurrence without the periodicity modulo 3 is also observed in the RNA coding genes (ribosomal, transfer, and small nuclear RNA genes) and in the noncoding genes (introns and 5' regions of eukaryotic nuclei). However, there are two exceptions to this YRY(N)6YRY rule: the protein coding genes of eukaryotic nuclei, and prokaryotes, where YRY(N)6YRY has the second highest value after YRY(N)0YRY (YRYYRY) with a periodicity modulo 3. When we go backward in time with the analytical expression, the protein coding genes of both eukaryotic nuclei and prokaryotes retrieve the YRY(N)6YRY preferential occurrence with a periodicity modulo 3 after 0.2 back transversions per base. In other words, the actual protein coding genes of chloroplasts and mitochondria are similar to the primitive protein coding genes of eukaryotic nuclei and prokaryotes. On the other hand, this application represents the first result concerning the mutation process in the model of DNA sequence evolution we recently proposed. According to this model, the actual genes on the R/Y alphabet derive from two successive evolutionary genetic processes: an independent mixing of a few nonrandom types of oligonucleotides leading to genes called primitive followed by a mutation process in these primitive genes.(ABSTRACT TRUNCATED AT 400 WORDS)

Base Sequence↗

PubMed1999

Clinical virology of rhinoviruses.

Rhinoviruses cause more infections in humans than any other micro-organism. These acid-sensitive picornaviruses infect epithelial cells following inoculation onto the nasal mucosa and are detected reliably in nasopharyngeal secretions. Rhinovirus colds occur year round, with a peak of illness in the fall. Type-specific serum antibody correlates with protection against infection. The fact that there are at least 100 different immunotypes makes development of an effective vaccine unlikely. Nasopharyngeal secretions must be sampled for detection of rhinovirus by culture or RT-PCR. Efficient isolation of virus requires inoculation into two different types of sensitive cell cultures (i.e., fibroblasts and HeLa cells). RT of conserved sequences in the 5' noncoding region of the viral RNA to produce cDNA for PCR amplification has been coupled with detection of amplimers either by gel electrophoresis after nested PCR or by hybridization with labeled oligonucleotide probes to detect one viral genome in samples. In two studies in which both RT-PCR and cell cultures were used, virtually all of the positives were identified with RT-PCR; culture in two cell lines identified 75-80% of the positives. In year-round surveillance, 50% of colds in adults and children were rhinovirus positive. The symptoms occurring during rhinovirus colds are caused by the host's response to the virus, not by the virus itself. Elaboration of cytokines by infected epithelial cells is central to symptom pathogenesis.

Common Cold↗

PubMed2006

RNAi: a potential therapy for the dominantly inherited nucleotide repeat diseases.

Genetic diseases that are accompanied by central nervous system involvement are often fatal. Among these are the autosomal dominant neurogenetic diseases caused by nucleotide repeat expansion. For example, Huntington's disease (HD) and spinal cerebellar ataxia are caused by expansion of a tract of CAGs encoding glutamine. In HD and the other CAG-repeat expansion diseases, the expansion is in the coding region. Myotonic dystrophy is caused by repeat expansions of CUG or CCTG in noncoding regions, and the mutant RNA is disease causing. Treatments for these disorders are limited to symptomatic intervention. RNA interference (RNAi), which is a method for inhibiting target gene expression, provides a unique tool for therapy by attacking the fundamental problem directly. In this review, we describe briefly several representative disorders and their respective molecular targets, and methods to accomplish therapeutic RNAi. Finally, we summarize studies performed to date.

Animals↗

PubMed2004

Circular box C/D RNAs in Pyrococcus furiosus.

Box C/D RNAs are small, noncoding RNAs that function in RNA modification in eukaryotes and archaea. Here, we report that box C/D RNAs exist in the rare biological form of RNA circles in the hyperthermophilic archaeon Pyrococcus furiosus. Northern analysis of box C/D RNAs reveals two prominent RNA species of different electrophoretic mobilities in total P. furiosus RNA preparations. Together, the results of Northern, ribozyme, RT-PCR, and lariat debranching analyses indicate that the two species are circular and linear RNAs of similar length and abundance. It seems that most, if not all, species of box C/D RNAs exist as circles in P. furiosus. In addition, the circular RNAs are found in complexes with proteins required for box C/D RNA function. Our finding places box C/D RNAs among the extremely few circular RNAs known to exist in nature. Moreover, the unexpected discovery of circular box C/D RNAs points to the existence of a previously unrecognized biogenesis pathway for box C/D RNAs in archaea.

Animals↗

PubMed2001

Recombinant hepatitis E virus genomes infectious for primates: importance of capping and discovery of a cis-reactive element.

Hepatitis E virus recombinant genomes transcribed in vitro from two cDNA clones differing by two nucleotides were infectious for chimpanzees. However, one cDNA clone encoded a virus that was attenuated for chimpanzees and unable to infect rhesus monkeys. The second cDNA clone encoded a virus that infected both chimpanzees and rhesus monkeys and caused acute hepatitis in both. One mutation differentiating the two clones identified a cis-reactive element that appeared to overlap the 3' end of the capsid gene and part of the 3' noncoding region. Capping of the RNA transcripts was essential for infectivity.

Animals↗

PubMed2006

Epstein-Barr virus noncoding RNAs are confined to the nucleus, whereas their partner, the human La protein, undergoes nucleocytoplasmic shuttling.

The Epstein-Barr virus (EBV) noncoding RNAs, EBV-encoded RNA 1 (EBER1) and EBER2, are the most abundant viral transcripts in all types of latently infected human B cells, but their function remains unknown. We carried out heterokaryon assays using cells that endogenously produce EBERs to address their trafficking, as well as that of the La protein, because EBERs are quantitatively bound by La in vivo. Both in this assay and in oocyte microinjection assays, EBERs are confined to the nucleus, suggesting that their contribution to viral latency is purely nuclear. EBER1 does not bind exportin 5; therefore, it is unlikely to act by interfering with microRNA biogenesis. In contrast, La, which is a nuclear phosphoprotein, undergoes nucleocytoplasmic shuttling independent of the nuclear export protein Crm1. To ensure that small RNA shuttling can be detected in cells that are negative for EBER shuttling, we demonstrate the shuttling of U1 small nuclear RNA.

Active Transport, Cell Nucleus↗

PubMed2003

Accumulation of mitochondrial DNA mutations in human immunodeficiency virus-infected patients treated with nucleoside-analogue reverse-transcriptase inhibitors.

Nucleoside reverse-transcriptase inhibitor (NRTI) therapy for human immunodeficiency virus (HIV) infection has been associated with mitochondrial DNA (mtDNA) polymerase-gamma inhibition and subsequent mtDNA depletion. Effects on mtDNA mutation, although suggested by critical involvement of polymerase-gamma in DNA-repair reactions, are unknown. In the present study, we assessed the nature and frequency of mitochondrial genome sequence differences in peripheral-blood samples taken prior to NRTI therapy and after 6-77 mo of treatment in 16 NRTI-treated patients. Samples from 10 HIV-infected, treatment-naive control individuals were taken at similar time intervals. Single-stranded conformation polymorphism (SSCP) and DNA-sequencing analysis techniques were used to detect mitochondrial genome sequence variants between paired longitudinal samples, and heteroplasmic populations were quantified after cloning and repeat SSCP/sequencing. Of 16 individuals treated with NRTIs, 5 exhibited altered SSCP profiles associated with the development of novel heteroplasmic DNA sequence changes, whereas no SSCP pattern change within these regions was observed in the control individuals. Heteroplasmic sequence changes were distributed across four regions of the genome: the noncoding region to 12S ribosomal RNA, reduced-nicotinamide-adenine-dinucleotide dehydrogenase 1, and cytochrome oxidase subunits I and III. Of the total of 26 patients who were examined in the present study, 4 of 5 patients with detectable mtDNA sequence changes since commencement of therapy developed evidence of peripheral fat wasting (lipoatrophy) between sample intervals (P=.031). One patient, without detectable sequence changes on NRTI therapy, also developed lipoatrophy. Levels of mtDNA copies/cell in blood samples were determined by quantitative PCR for 11 of the 16 NRTI-exposed patients; 7 of these 11 patients showed reduced levels of mtDNA in blood after therapy, including all 3 patients tested with evidence of mtDNA sequence changes on therapy. These data indicate that NRTI therapy provides conditions permissive for the development of peripheral-blood mtDNA mutations in vivo.

Base Sequence↗

PubMed1988

Expression of a histone H1-like protein is restricted to early Xenopus development.

Genes whose expression is restricted to oogenesis and early development may have important functions in these processes. Northern analysis showed that Xenopus B4 mRNA is expressed in oogenesis and embryogenesis through to the neurula stage. Immunocytochemistry with anti-B4 antibodies showed that B4 protein is only detectable in preneurula stages; it is localized to nuclei and is associated with metaphase chromosomes. Immunoblotting revealed approximately constant levels of B4 protein per embryo for the first 2 days of development. Thus, as the number of nuclei increases during early development, the amount of B4 protein per nucleus is diluted out. Sequencing of two B4 cDNA clones revealed that the predicted B4 translation product is a 29-kD protein with 29% identity with histone H1, distributed over the entire length of its sequence. The B4 protein also has certain other H1 protein characteristics--a tripartite structure consisting of a mainly hydrophobic central domain flanked by an amino-terminal segment and a long hydrophilic carboxyterminal tail containing a tandemly repeated amino acid motif. However, in contrast to histone H1 mRNA, B4 mRNA has a classic polyadenylation signal, is polyadenylated, and lacks the histone H1 3' noncoding consensus sequence involved in RNA processing.

Amino Acid Sequence↗

PubMed1991

Overlapping retrovirus U5 sequence elements are required for efficient integration and initiation of reverse transcription.

A secondary structure in the 5' noncoding region of avian retrovirus RNA, called the U5-leader stem, was shown previously to have a role in initiation of reverse transcription (D. Cobrinik, L. Soskey, and J. Leis, J. Virol. 62:3622-3630, 1988). We now show that an additional RNA secondary structure near the U5 terminus, called the U5-IR stem, is also important for reverse transcription. Mutations that disrupt the U5-IR stem cause a replication defect associated with both a decrease in synthesis of viral DNA in infected cells and a decrease in initiation of reverse transcription in melittin-permeabilized virions. Structure-compensating base substitutions in the U5-IR restore reverse transcription efficiency. In viral DNA, U5-IR sequences are included in the U5 terminal region that functions as a viral integration donor site. When base substitutions are introduced into these sequences, a reduced efficiency of integration in vitro and in vivo is observed. These observations indicate that U5-IR sequences have a structural role in reverse transcription of viral RNA and a sequence-specific role in the integration of viral DNA.

Avian Sarcoma Viruses↗